Tail regeneration reduction in lizards after repetitive amputation or cauterization reflects an increase of immune cells in blastemas

2018·27.2 MB·

by  Alibardi, Lorenzo

ZOOLOGICAL RESEARCH
Tail regeneration reduction in lizards after repetitive
amputation or cauterization reflects an increase of
immune cells in blastemas
LorenzoAlibardi1,*
1ComparativeHistolabPadua,DepartmentofBiology,UniversityofBologna,Bologna40126,Italy
ABSTRACT INTRODUCTION
The regenerating tail of lizards is an outstanding and unique
Lizards are key amniote models for studying
case of massive organ regeneration in amniotes, i.e., fully
organ regeneration. During tail regeneration in
terrestrial vertebrates (Alibardi, 2010a; Bellairs & Bryant, 1985;
lizards, blastemas contain sparse granulocytes,
Fisher et al., 2012; Gilbert et al., 2013). The process of tail
macrophages,andlymphocytesamongtheprevalent regeneration in lizards is a uncommon event given the lack of
mesenchymal cells. Using transmission electron anyorganregenerationinotheramniotes. Duringregeneration,
microscopytoexaminescarringblastemasafterthird numeroustissuesarereformedandcombinedintoafunctional
tail, albeit simplified in comparison to the original. Therefore,
and fourth sequential tail amputations, the number
lizards are critical vertebrates for analyzing successful versus
of granulocytes, macrophages, and lymphocytes
unsuccessful tissue and organ regeneration in amniotes, with
increased at 3–4 weeks in comparison to the
potential consequences for mammalian regeneration (Alibardi,
first regeneration. An increase in granulocytes and 2010a, 2014; Gilbert et al., 2013; Lozito & Tuan, 2017).
agranulocytes also occurred within a week after Rare cases of anomalous regeneration have been recorded
blastemacauterizationduringtheprocessofscarring. in relation to parasitic infection resulting in impaired health
conditions (Oppliger & Clobert, 1997) or following intense
Bloodatthethirdandfourthregenerationalsoshowed
damage accompanied by severe inflammation (Baffoni, 1950).
a significant increase in white blood cells compared
Tailregenerationcanbealteredorinhibitedunderexperimental
with that under normal conditions and at the first manipulationssuchascauterization,woundingtheregenerating
regeneration. Theextracellularmatrixofthescarring blastema, or removing the apical epidermal peg, thus resulting
blastema, especially after cauterization, was denser in scarring (Alibardi, 2010a, 2013, 2014). Therefore, normal
regeneration in lizards depends on processes that control
than that in the normal blastema and numerous
inflammationbutcanbeinterruptedbytheabovetreatments.
white blood cells and fibroblasts were surrounded
Inflammationisatypicalcellandhumoralresponseelicited
by electron-pale, fine fibrinoid material mixed with
bytraumaticmechanical(cuts,amputation),chemical(irritants,
variablecollagenfibrils. Inadditiontopreviousstudies, toxins), or physical (heat, cold, radiation) stimuli to tissues.
thepresentobservationssupportthehypothesisthat Inflammation limits tissue and organ plasticity in different
an increase in inflammation and immune reactions models of vertebrates (Leavitt et al., 2016; Mescher et al.,
2013; Montali, 1988). Invasion of the regenerating tail
determine scarring rather than regeneration. These
blastema from immune cells, granulocytes, macrophages,
new findings verify that an immune reaction against
and lymphocytes is poorly known in lizards, and based
mesenchymalandepidermalcellsoftheregenerative
only on qualitative observations (Alibardi, 1994, 2010b, 2013,
blastema isoneofthemaincausesforthefailureof 2016; Alibardi & Sala, 1988). These previous ultrastructural
organregenerationinamniotes.
Keywords:Lizard;Tail;Repetitiveregeneration;Blood; Received:02January2018;Accepted:20June2018;Online:06July2018
Immunecells;Ultrastructure *Correspondingauthor,E-mail:[email protected]
DOI:10.24272/j.issn.2095-8137.2018.050
SciencePress ZoologicalResearch39(6):413–423,2018 413
and immunohistochemical studies have indicated that the mminlengthandconiforminshape,itwascutatthebaseand
scarring limb undergoes strong invasion from hematogenous the blastema with extravasating blood was collected as above
cells, in particular mononucleate cells (macrophages and fromthefiveanimals,representingblastemaandbloodsamples
lymphocytes). of the first regeneration. The operation was repeated in the
The importance of clarifying the role of the immune system following weeks to collect blastema and blood samples of the
in regenerative plasticity in amniotes was confirmed recently second,third,andfourthregeneration.Ascontrols,threelizards
based on a transcriptome study comparing the regenerating were left to regenerate their tails normally. All outgrowths and
tail blastema to that present in scarring limbs of the same collectedbloodsampleswerefixedasabove, dehydrated, and
lizards (Vitulo et al., 2017). The study indicated that embeddedinLowcrylresinunderUVexposurefor2dat4°C.
inflammation-elicitinggeneswereunchangedbutimmune-genes
Tailcauterization
were strongly down-regulated in the tail blastema, whereas
inflammatory genes were up-regulated but immune-genes Tail cauterization was performed on three lizards with
remainedunchangedinthelimbofthesameanimals. regenerating blastemas 2–3 mm in length. Using a hot plate,
alaminarspatulawasheatedfor10s,thenappliedthreetimes
Theregeneratingblastemathereforeappearstofunctionas
for1–2stothetipoftheblastema,scoldingitwithoutapparent
an immune-privileged/suppressed organ, like the developing
externaldamagetotheskin(i.e.,nobleedingorcutting). The
tail or limb, and the presence of immune cells under normal
animals were left for 5–6 d in a cage, with the blastema then
tail regeneration is likely limited and does not influence the
collected and fixed after growing ceased. The cauterized
process. Previous electrophoretic and hematological studies
blastemas were cut in half, fixed in 4% paraformaldehyde as
have indicated that the number of lymphocytes/macrophages
aboveanddehydratedinethanol;onehalfwasthenembedded
and the gamma-globulin fraction tend to increase when tail
inwax(forotherstudies)andtheotherhalfwasembeddedin
regenerationisreducedorinhibited(Alibardi,2014). However,
plasticandtransparentresinLowcryl.
the specific production of anti-regenerative antibodies or
lymphocytes has not been demonstrated, and therefore the
Preparationforhistologyandelectronmicroscopy
increaseinthenumberofimmunecellsandgammaglobulins
Lowcryl-embedded blastemas were sectioned along the
might simply reflect a general state of inflammation. To date,
longitudinalplaneusinganultramicrotomeandwerecollected
no studies have measured the number or type of white blood
on glass slides, stained with 1% toluidine blue, and observed
cells, especially macrophages and lymphocytes, invading the
under a light microscope for histological analysis. The
blastema and circulating in the blood during manipulations
embedded blood samples (pellets) were also sectioned with
that stimulate scarring (Alibardi, 2013, 2014). The present
an ultramicrotome. From regions of interest, thin 50–90-nm
ultrastructural study aimed to investigate the hypothesis that
sections were collected on copper grids for transmission
the significant increase in white blood cells, in particular
electron microscopy (TEM) study. The sections were stained
macrophagesandlymphocytes,likelyexertsanimmunological
for45minin1%uranylacetatedwatersolutionandfor5min
reaction against blastema cells induced to form scarring
in lead citrate according to routine methods, then observed
fibroblastsinsteadofamesenchymalmassofcells.
under a Zeiss C10 electron microscope operating at 60 kv.
MATERIALSANDMETHODS Images were acquired using a digital camera incorporated in
theelectronmicroscope.
Repetitivetailamputations
Tworegenerativeblastemas(3mminlength)wereprepared
Five adult wall lizards (Podarcis muralis) of both sexes were for the Gomori reaction to reveal acid phosphatases, typical
utilized in the present study; they all survived and were markersusedtoidentifylysosomegranulesinwhitebloodcells
eventuallyreleasedinthewild.AllexperimentsfollowedItalian (Troyer, 1980), under electron microscopy. Tissues were fixed
regulations on animal care and handling (art. 5, DL 116/92). at 0–4°C in 0.25% glutaraldehyde and 4% paraformaldehyde
Thelizardswereleftfor20minat4–6°Ctoinducehypothermic in 0.12 mol/L phosphate buffer at pH 7.4. The tissues were
numbness, and the tail was amputated with a razor blade at then cut with a vibratome to collect 50–70-µm sections, which
aboutthesecondtothirdproximal. Thesurfaceofthestump wereincubatedfor45minin0.5mol/LacetatebufferatpH4.8
was kept wet and warm with the application of a small drop andtheninGomori-leadmediumfor90minat27–30°Cforthe
of Ringer fluid, and the extravasating blood from the stump detectionofacidphosphatases(Troyer,1980). Afterwashingin
wascollectedwithaPasteur’spipette(30–50µL)intoasmall acetate buffer for 30 min, the sections were rinsed in distilled
0.5-mLEppendorfvialuntilnofurtherbleedingwasobserved. waterfor5min, post-fixedin1%osmiumtetroxidefor1h, and
Afixativeconsistingof0.2mLof4%paraformaldehydesolution then dehydrated and embedded in EPON resin. Thin sections
in0.1mol/LphosphatebufferatpH7.4wasrapidlyaddedtothe obtainedusinganultramicrotomewerecollectedoncoppergrids
bloodsamplesfor5min. andobservedunderaZeissC10electronmicroscopeasabove.
The Eppendorf vials were centrifuged for 5 min at 10 000
r/min to form a pellet, and fixation lasted about 8 h. From Cell counts during successive amputations and after
the same lizards, a 2-mm portion of the normal stump of the cauterization
detachedtailwasfixedinthesamefixativetorepresentnormal Basedontherelativepercentagesofcellsindifferentstagesof
controls. Whentheblastemaofthefirstregenerationwas2–3 regeneration,twoapproacheswereusedtocomparetherelative
414 www.zoores.ac.cn
variations in white blood cells present in blastemas or in blood lysosomes were acid phosphatase-positive (Figures 1G, 2A),
following repetitive amputations of the tail or its cauterization. and in the granulocytes some granules appeared positive for
Firstly, the numbers of white blood cells (granulocytes and theenzymeaswell(Figure2B).Amongthefibroblastspresent
agranulocytes) were counted from whole TEM thin sections intheoutgrowth,numerouscollagenfibrilswereseen,andthe
of regenerative blastemas at the first and fourth regeneration, extracellular spaces were reduced to form dense connective
and in cauterized blastemas. The number of detected white tissueswithrespecttothatofthenormalblastema(Figure2C).
blood cells in each case was indicated as a percentage out of Furthermore, lymphocytes appeared relatively frequently
400–500blastema(mesenchymal)cellscountedinblastemasat among cells of the blastema in outgrowths at the fourth
thefirstandfourthregenerationandoneweekaftercauterization. regeneration (Figures 3, 4). Cells recognized as lymphocytes
Secondly,thebloodsampleswereexaminedandthenumberof were smaller than blastema cells and showed a more
whitebloodcells,indicatedasapercentageoutof700–900red electron-dense cytoplasm compared with the fibroblasts, with
blood cells, was counted in the blood pellet sections collected variable perinuclear chromatin. Large cytoplasmic regions of
fromlizardsinnormal,first,third,andfourthregenerations. The the lymphocytes contacted the membrane of the blastema
number of TEM sections (n) for each individual, as well as the cells (Figure 3A–C) or occurred through their thin elongation
mean,standarddeviation(mean±SD),andstatisticalsignificance (Figure 3D, E). In some cases, the activated lymphocytes
usingone-wayANOVAcomparingvariationsindifferentstages, presented a very irregular surface with typical filopodia, as
are reported in the text. Statistical significance was set at *: in the macrophages (Figure 4A). In these cases, their exact
P<0.05,**:P<0.01,or***:P<0.001. identification was doubtful, and therefore in our quantitative
counts we considered these cells collectively as agranulocytes
RESULTS
(seeFigure5). Weconsideredtruemacrophagesonlyforlarger
cells exceeding 10 µm. Other cells with a paler and vesicular
Grossandgeneralmicroscopyobservations
cytoplasm, but containing large phagosomes or cell remnants,
The three lizards allowed to regenerate their tails normally
were considered as dying phagocytes (Figure 4B). The more
produced long and scaled regenerated tails in about 30 d
frequent granulocytes encountered among the connective
following amputation (Figure 1A). Conversely, compared with
tissues of the outgrowths were represented by basophilic cells.
first and second regeneration blastemas, some third and most
These cells were recognized by their numerous and large
fourth regeneration blastemas appeared as short and hard
globulargranulesandbytheirroundish,non-polymorphicnuclei,
scarringoutgrowths.Theseoutgrowthsshowedirregularscalation
as observed for neutrophilic granulocytes (Figure 4C). These
patternsandwere2–3mminlength(insetinFigure1A).
cellswereinclosecontactwiththeblastemacells,althoughsome
The normal regenerating tail blastema contained a
amorphousmaterialandcollagenfibrilswereoftenseenbetween
homogenous mass of mesenchymal cells and fibroblasts
thetwocelltypes(Figure4D).Smallvesicles(0.1–0.2µminsize)
localized underneath a multilayered wound (regenerating)
were alsoseen inthese regions, thoughtheir originsremained
epidermis (Figure 1B). Electron microscopy showed that these
unclearfromourstaticimages.
fibroblast-mesenchymal cells possessed a variable number of
Thenumberofwhitebloodcellspresentinfirstregeneration
narrowendoplasmiccisternaeandeuchromaticnucleiwithlarge
blastemas, out of 400–500 blastema cells, compared to the
nucleoli. The cells were surrounded by a loose extracellular
number of white blood cells in fourth regeneration blastemas,
space containing sparse amorphous material (hyaluronic acid)
ispresentedinFigure5. Resultsshowedanalmostthree-fold
andscarceunbandedcollagenfibrils(Figure1C).
increaseinwhitebloodcellsatthefourthregeneration(mean
The inner tissues present in the regenerating outgrowths
15.9), especially agranulocytes, compared to that at the first
at the fourth regeneration displayed less regular connective
regeneration(mean5.0). Thisvaluewassignificantaccording
tissue and irregular bundles of extracellular (collagenous)
totheANOVAtest(Table1,Figure5).
fibrils located among fusiform fibroblasts, especially in the
dermis(Figure1D).Incauterizedblastemas,extracellularfibrils Electron microscopy and count variations in cauterized
or masses of fibrin material were present among fusiform outgrowths
fibroblastsandbloodvessels(Figure1E). After cauterization, the blastema stopped its growth. Among
blastemacells(fibroblasts),areducedextracellularmatrixwas
Electron microscopy and count variations at first and present in comparison to the normal blastema (Figure 6A, B).
fourthregenerations White blood cells were more frequently encountered than in
The normal blastema (first regeneration) contained loose thenormalblastemaatthefirstregenerationandappearedas
connective tissue with many mesenchymal cells and sparse agranulocytes (macrophages-lymphocytes) and granulocytes
macrophages (Figure 1C, F). The Gomori reaction showed withlargegranules,likelybasophils. Quantificationindicateda
that macrophages in apical or more proximal regions of three-foldincrease(mean15.1,seeFigure5)inwhitehematic
the blastema contained acid phosphatase positive lysosomes cells in the blastema at 6 d post-cauterization compared with
(Figures 1G, 2A). In the regenerating outgrowths following the number of white cells present in the blastemas in the
thefourthregeneration,electronmicroscopyrevealedfrequent first regeneration (mean 5.0, see Figure 5). This value was
macrophages and granulocytes containing lysosomes in significantaccordingtotheANOVAtest(Table1,Figure5).
various stages of digestion (Figure 1F). In the macrophages,
ZoologicalResearch39(6):413–423,2018 415
Figure1Grossaspectsofregeneratedtailversusscarringtailoutgrowth(A)andmicroscopyimagesofareasoftheregenerating
Figure 1 Gross aspects of regenerated tail versus scarring tail outgrowth (A) and microscopy
tail(B–G)
A:Largelysicmaleadg,eresg eonfe ararteedasta oilfa tth30e drepgoset-naemrpauttiantigon t.aAilrr o(wBh–eGad)i ndicatestransitionpointwithstumpoforiginaltail. Scalebar: 1mm. Insetshowsa
tailoutgrowthatfourthregeneration(arrowheadonamputationregion). Scalebar: 0.5mm. B:Histologyofafirstregenerationblastema. Scalebar: 10µm.
A: Largely scaled, regenerated tail at 30 d post-amputation. Arrowhead indicates transition point
C:Electronmicroscopyviewoffirstregenerationblastemacells. Arrowsindicatecollagenousfibrilswithamorphousmatrix. Scalebar: 0.5µm. D:Histology
with stump of original tail. Scale bar: 1 mm. Inset shows a tail outgrowth at fourth regeneration
ofafourthregenerationblastema.Scalebar:10µm.E:Histologyofablastemaat6dpost-cauterization,showinglargemassesofpaleextracellularmaterial
(arrows)und(earrnreoawthhweoaundd eopnid earmmisp.uStcaatleiobnar :r1e0gµiomn.)F. :MSaccarloep habgaerw: ith0.n5u mmeromus. sBec:o nHdairsytloysloosgoym esof(a rrao wfhierasdts )raengdefinloeproadtiiao(nar rows)withina
firstregenerationblastema.Scalebar:1µm.G:Macrophagewithintheapicalregionofafirstregenerationblastemawithacidphosphatasepositivelysosomes
blastema. Scale bar: 10 μm. C: Electron microscopy view of first regeneration blastema cells.
(arrows).Scalebar:1µm.de:dermis;ex:extracellularmatrix;n:nucleus;w:woundepidermis.
13
416 www.zoores.ac.cn
Figure 2 Electron microscopy view of white blood cells in a first regeneration blastema (A, B) and a fourth regeneration
Figure 2 Electron microscopy view of white blood cells in a first regeneration blastema (A, B) and
fibroblast(C)
A:Maacr ofpohuagretshw ritehgaecindeprhaostpiohanta sfeibproosibtivleagsrta n(uCle)s (arrows)inmoreproximalareaoftheblastema. Scalebar: 0.5µm. B:Granulocytewithsomeacid
phosphatasepositivegranules(arrows).Scalebar:0.25µm.C:Fibroblastincontactwithlargebundleofcollagenfibrils.Scalebar:1µm.col:collagenfibrils;
A: Macrophages with acid phosphatase positive granules (arrows) in more proximal area of the
ex:extracellularmatrix;n:nucleus;ns:non-specificgranules.
blastema. Scale bar: 0.5 μm. B: Granulocyte with some acid phosphatase positive granules
ZoologicalResearch39(6):413–423,2018 417
15
(arrows). Scale bar: 0.25 μm. C: Fibroblast in contact with large bundle of collagen fibrils. Scale
bar: 1 μm. col: collagen fibrils; ex: extracellular matrix; n: nucleus; ns: non-specific granules.

Figure3Whitebloodcellspresentinafourthregenerationblastema
Figure 3 White blood cells present in a fourth regeneration blastema
A:Mesenchymalfibroblastclosetodarkeragranulocyte(arrow). Scalebar:1µm. B:Largeappositionalarea(arrowheads)betweenlymphocyte(darker)and
blastemacells(palerfibroblast).Scalebar:0.25µm.C:Contactareawithfilopodia(arrows)localizedbetweenalymphocyteandfibroblast.Scalebar:0.25µm.
D:Macrophagefilopodcontacting(arrow)mesenchymalcell. Scalebar: 0.25µm. E:Filopodfromamacrophagecontactingorinvading(arrows)ablastema
cell.Arrowheadspointtoseeminglyextracellularvesicles.Scalebar:0.25µm.ex:extracellularmatrix;ly:lymphocyte;ma:macrophage;n:nucleus.
16
A pale and finely granular material, resembling a filaments(Odland&Ross,1968), werealsoembeddedinthe
plasma-fibrinous exudate, was present in the extracellular exudate. These fibrils were irregularly distributed within the
matrix (Figure 6C–E). Denser coarse filaments or fibrils of extracellularmatrix.
30–40 nm and of unknown nature but resembling fibrin
418 www.zoores.ac.cn
Figure4Electronmicroscopyviewofwhitebloodcellswithinafourthregenerationblastema
Figure 4 Electron microscopy view of white blood cells within a fourth regeneration blastema.
A:Agranularelectron-densecellwithfilopodia(arrows)andcytoplasmicdensebodies(arrowheads,likelylysosomes). Scalebar:1µm. B:Palecellwithtwo
largephagosoAm:e sA(agrrorawnheualdasr) neealercatcreolnlw-dithenfilsoep odciae(lal rrowwsi)t.hS cfailelobpaor:d1iaµ m(.aCrr:oBwasso)p hilagnrdan ulcoycyttoep(alarrsomwsicsh odwelanrsgee debnosdeigeras nules)detected
amongblastemacells. Scalebar: 1µm. D:Narrowextracellularspacepresentbetweenabasophilandblastemacell,wherecollagenfibrilsandamorphous
(arrowheads, likely lysosomes). Scale bar: 1 μm. B: Pale cell with two large phagosomes
materialarepresent(arrows). Arrowheadspointtoapparentextracellularvesicles. Scalebar:0.25µm. bl:blastema(mesenchymal)cell;col:collagenfibrils;
ex:extracellul(aarrsrpoawceh;ne:andusc)le nuse.ar a cell with filopodia (arrows). Scale bar: 1 μm. C: Basophil granulocyte (arrows
show large dense granules) detected among blastema cells. Scale bar: 1 μm. D: Narrow
Electron microscopy of blood cells in normal, first, third, regeneration)(Table2,Figure7).Thevaluewasnotsignificant
andfourtherxetgraecneelrlautliaorn space present between a basophil andb etbwlaesetnemnoar mcaellla, nwdhfiersrte recgoellnaegreantio nfiborrilnso ramnadl and second
The blood cells derived from pellets of normal blood samples regeneration but was significant between first and second
andafterthefirst,third,andfourthregenerationdemonstrated regeneration versus fourth regeneration and highly significant
a significan t 3–4-fold increase in white blood cells in general betweennormalandfourthregeneration(Figure178).
(1.2% in normal blood versus 5.1% in blood at the fourth
ZoologicalResearch39(6):413–423,2018 419
25 White blood cells in both pellets and blood vessels
in the blastema were mainly represented by granulocytes
20
(especiallybasophils), thoughmacrophagesandlymphocytes
%)
C (15 were also seen (Figure 8A). Numerous cells were likely
B
BC/10 lymphoblasts, characterized by a round shape and pale
W
cytoplasm, and containing sparse free ribosomes and a
5
nucleus featuring a more diffuse (unpacked) chromatin than
0 that of small lymphocytes, macrophages, or granulocytes
1stR 4thR Caut
(Figure 8B–E). Completely differentiated plasma-cells were
Figure 5 Statistical values and significance of variations in
only found occasionally in blood pellets sections and within
white blood cells/blastema cells in first regeneration (1stR)
vesselsintheblastemaorinscarringoutgrowths(Figure8F).
comparedtofourthregeneration(4thR)orcauterized(Caut)
**:P<0.01;***:P<0.001.

Figure6Electronmicroscopyviewofblastema6daftercauterization
Figure 6 Electron microscopy view of blastema 6 d after cauterization.
A:Basophilgranulocytes(arrow)andmacrophages(arrowhead)presentamongblastemacells.Scalebar:2µm.B:Electron-paleextracellularmatrixpresent
A: Basophil granulocytes (arrow) and macrophages (arrowhead) present among blastema cells.
amongblastemacells. Scalebar: 1µm. C:Darklymphocytewithintheelectron-paleextracellularexudate. Scalebar: 1µm. D:Denseamorphousmaterial
presentwithintheextracSecllaullea rbpaar:le 2e xμumda. tBe.: EScleacletrobanr-:pa0l.e2 5exµtmra.ceEl:luDlaern smeratfirbixri llparremseantet raiaml(oanrrgo wbsla)setemmbead cdeeldlsi.n Spcaalleer bexaur:d a1t ebetweentwodarkcells
(arrowheads).Scalebarμ:0m.2. Cµ:m D.balr:kb llaysmtepmhaoccyeltles ;wdsitmh:ind ethnes eeleexctrtarocenl-luplaalrea enxdtlraarcgeelllyualamr oerxpuhoduastem. Satcearliael ;beaxr:: e1x tμramce. lDlu:la Drsepnascee ;n:nucleus.
amorphous material present within the extracellular pale exudate. Scale bar: 0.25 μm. E: Denser
420 www.zoores.ac.cn
20
DISCUSSION Zimmermanetal.,2010).Despitetheseshortcomings,thepresent
study clearly showed that significant increases in the number of
This ultrastructural study demonstrated a significant increase
granulocytes, lymphocytes, and particularly macrophages in the
in white blood cells, about three times higher than that
blastemaandbloodwereassociatedwithscarring.
observed during the first regeneration, in the cauterized and
fourth repetitive regeneration blastemas, confirming previous
studies (Alibardi, 2010a, 2010b, 2013, 2014). In particular, 8
mononucleate macrophages or activated lymphocytes were
predominant over other blood cell types (Figure 5), whereas %) 6
granulocytes were more abundant than agranulated cells in C (
B
R 4
blood(Figure7). Woundexudate,whichcontainedfibrin,was C/
B
abundant in the cauterized blastemas, likely due to plasma W
2
extravasation following cauterization. The removal of the
exudatelikelygeneratedintenseinflammationintheblastema,
0
leadingtoscarring(Alibardi,2013). NB 1stR 3rdR 4thR
Figure 7 Statistical values (ANOVA test) and significance of
Table1Percentagesofwhitebloodcellsinfirstregenerative
variations in white blood cells/erythrocytes in normal blood
blastemacomparedwiththosepresentinfourthregenerative
(NB), first regeneration (1stR), third regeneration (3rdR), and
blastemaand6dafterblastemacauterization
fourthregeneration(4thR)
1streg 4threg Cauterized
**:P<0.01;***:P<0.001.
n 5 4 3
The present study indicated that an inflammatory reaction
G(%) 45.2 28.8 40.2
involving an average of 15% of white blood cells invading the
A(%) 54.8 71.2 59.8
blastemawasstrongenoughtolimitorimpairtailregeneration
WBC(%) 5.0±1.5 15.9±1.6 15.1±1.8
in lizards. Therefore, it is not surprising that the healing
n: Numberofsamples. G:Granulocytes(heterophils-neutrophils, limb of Podarcis muralis at 11–12 d of regeneration after
basophils, and eosinophils). A: Agranulocytes (monocytes and amputation, where inflammation includes over 50% of white
blood cells in comparison to the total cell types present
lymphocytes).WBC:Whitebloodcells.
in a forming blastema (Alibardi, 2010b, 2016; 50.5%±2.4%
Table2Percentagesofwhitebloodcellsinthebloodofnormal in three quantified cases), gives rise to a scar. The
lizardsandinfirst,third,andfourthregeneration main proportion of immune cells, about 70% of the total,
consisted of macrophages/lymphocytes of difficult specific
Normal 1streg 3threg 4threg
identification. The increase in basophils/eosinophils and
n 3 4 3 3
macrophages/lymphocytes detected in the present study in
G(%) 56.7 56.2 58.0 54.9
scarring tails indicated that these cells were likely associated
A(%) 43.3 43.8 42.0 45.1
with the formation of scarring connective tissue. However,
WBC(%) 1.2±0.45 2.1±0.41 2.5±0.37 5.1±0.53
direct stimulation of myofibroblasts and fibrocytes in lizards
n: Numberofsamples. G:Granulocytes(heterophils-neutrophils, from these cells was not shown. The differentiation of the
lattercellscangiverisetoascarringblastemaduringthefourth
basophils, and eosinophils). A: Agranulocytes (monocytes and
regenerationinacauterizedtailstump(Alibardi,2010a,2013)
lymphocytes).WBC:Whitebloodcells.
orinthecauterizedblastema(presentstudy).
Most lymphocytes contained sparse ribosomes and lacked Numerous studies on mammalian and non-mammalian
endoplasmic reticulum vesicles, suggesting they were T-cells vertebrates have shown that inflammation, especially chronic
(Janossyetal.,1972). However,duetosomeuncertaintyinthe inflammation, is detrimental to regeneration (Ferguson &
discriminationbetweenmacrophagesandactivatedlymphocytes O’Kane, 2004; Leavitt et al., 2016; Mescher et al., 2013).
or between activated basophil and eosinophil granulocytes, the However, the type of injury can evocate different types of
presentquantitativedataonwhitecelltypesshouldbeconsidered inflammatorycells, inparticularmacrophages, someofwhich
with some caution. In the average sections of the blastema (healing or M2) actually favor regeneration in amphibians
or blood, cells appeared in their active, often motile condition, (Godwin & Rosenthal, 2014), fish (Petrie et al., 2014), and
and their shape and surface were distorted and not optimal for mammals(Heskethetal.,2017;Simkinetal.,2017). Thebest
unbiased morphological identification. Furthermore, the plane regeneratorsamongvertebrates,urodeleamphibians,andalso
of the randomly cut sections may also have intercepted cells many fish, possess low inflammation and immune responses
in a cytoplasmic or nuclear region that were tangential to the afterinjury,supportingthehypothesisthatimmuneefficiencyis
maincellulararea,makingidentificationmoreproblematic. Some themainobstacletoorganregenerationinamniotes. Another
studieshavealsoindicatedthatlymphocytesinfish,amphibians, clear indication of the negative effect of the immune system
and some reptiles possess active phagocytosis, making the onorganregenerationispresentinanuranamphibians,which
distinction with macrophages even more difficult (Li et al., 2006; aremoreterrestrially-adaptedthanmanyurodeles(Mescheret
ZoologicalResearch39(6):413–423,2018 421
al.,2013, 2017). Aftermetamorphosis, theimmuneresponse the type of macrophages and lymphocytes activated are
is enhanced, and the regeneration capability is lost in frogs, pro-inflammatory(M1),leadingtoscarformation.
toads,andsometerrestrialsalamanders(Mescheretal.,2013, The present quantitative ultrastructural study supports the
2017; Scadding, 1977, 1981). Evolution and potentiation of hypothesis that the immune system is likely the main cause
the adaptive immune system are correlated with the almost of failure of tissue and organ regeneration in lizards, and in
completelossoforganregenerationinendothermicamniotes. amniotesingeneral(Alibardi,2014). Futurefollow-upstudies
Thestronginflammationelicitedinbothavianandmammalian onthistopicarerequiredtodemonstratewhetherinflammatory
organs after injury leads to rapid resolution and variable cellsrespondtogeneralstimulationafterintenseandpersistent
degreeofscarring(Ferguson&O’Kane,2004; Heskethetal., tissue damage or if macrophages and lymphocytes can
2017). Itislikelythatafterlimbamputationorcauterizationin specificallyattachtomesenchymal,ependymal,andepidermal
lizards,traumaticeventsthatinduceconsiderableinflammation, cellsoftheregeneratingblastema.

Figure8Electronmicroscopyviewofbloodcellspresentinbloodvesselswithinablastema(A,D–F)orisolatedinbloodpellets
Figure 8 Electron microscopy view of blood cells present in blood vessels within a blastema (A,
(B,C)atthefourthregeneration
D–F) or isolated in blood pellets (B, C) at the fourth regeneration
A:Inthislargecapillary,mostlymphocytes(arrowheads),lymphoblasts(arrows),andmonocytes(doublearrows)areseen.Scalebar:2µm.B:Palelymphoblast
A: In this large capillary, most lymphocytes (arrowheads), lymphoblasts (arrows), and monocytes
detectedamongerythrocytes.Scalebar:1µm.C:Lymphoblastpresentamongerythrocytes.Scalebar:1µm.D:Polymorphonucleategranulocyte(arrowheads
(double arrows) are seen. Scale bar: 2 μm. B: Pale lymphoblast detected among erythrocytes.
indicatethreelobesofthenucleus).Scalebar:1µm.E:Mastocyte(basophil?)withfilopodia(arrows).Scalebar:1µm.F:Plasmacellwithinblastema.Scale
bar:2µm.col:collagenSbucnadlele ;benard:: e1n doμthme.li alCce: ll;Leyx:mepxhtroabcelallsutl arpmreastreixn;t eraym:eornytgh roecryyteth;rno:cnyutcelse.u sS;ncsa:len onb-asrp:e c1ifi cμgrman. uleDs:; pla:plasma-cell.
22
422 www.zoores.ac.cn